Inhibit Tumor Promotion Research Articles

Differential Effects of Flavonoid Compounds on Tumor Promoter-Induced Activation of the Human CYP1A2 Enhancer

Flavonoids, a family of naturally occurring polyphenolic compounds found in many fruits, nuts, vegetables, and beverages, appear to inhibit tumor promotion as part of their chemopreventive properties. To investigate at the molecular level the ability of flavonoids to inhibit tumor-promoting activity, we developed a cell line designed to screen for flavonoids that block the tumor promoter-mediated induction of activator protein-1 (AP-1) transcriptional activity. This cell line, T2Luc, is a HepG2-derived cell line stably integrated with a region of the human CYP1A2 5′-flanking gene containing two AP-1 binding sites linked to the thymidine kinase promoter-driven firefly luciferase reporter gene. Treatment of T2Luc with a commercial extract of green tea alone had no effect on luciferase activity, but did block the induction of luciferase when cells were further challenged with the tumor promoter phorbol 12-O-tetradecanoate 13-acetate (TPA). In contrast, treatment of cells with the flavonoid quercetin alone activated luciferase activity in a concentration-dependent manner and enhanced the TPA-induced transcription of luciferase. Gel mobility shift assays using nuclear extracts from cells treated with green tea extracts or TPA alone revealed induced binding of AP-1 proteins to the CYP1A2 3′AP-1 site. Pretreatment with green tea extracts did not inhibit the TPA-induced formation of AP-1 complexes. Quercetin treatment alone slightly enhanced binding of AP-1 complexes to this site. Our results suggest that these dietary chemopreventive agents may work through different pathways to modulate gene expression.

Induction of apoptosis by penta- O-galloyl-β- d-glucose through activation of caspase-3 in human leukemia HL-60 cells

Penta- O-galloyl-β- d-glucose is structurally related to (−)-epigallocatechin gallate and is isolated from hydrolyzed tannin. Penta- O-galloyl-β- d-glucose can inhibit tumor promotion by teleocidin. We investigated the effects of penta- O-galloyl-β- d-glucose and various tea polyphenols on cell viability in human leukemia HL-60 cells. In this study, we demonstrated that penta- O-galloyl-β- d-glucose was able to induce apoptosis in a concentration- and time-dependent manner; however, other polyphenols were less effective. We further investigated the molecular mechanisms of penta- O-galloyl-β- d-glucose-induced apoptosis. Treatment with penta- O-galloyl-β- d-glucose caused induction of caspase-3/CPP32 activity in dose- and time-dependent manner, but not caspase-1 activity, and induced the degradation of poly-(ADP-ribose) polymerase. Pretreatment with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) inhibited penta- O-galloyl-β- d-glucose-induced DNA fragmentation. Furthermore, treatment with penta- O-galloyl-β- d-glucose (50 μM) caused a rapid loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. Our results indicate that penta- O-galloyl-β- d-glucose allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA, and induces DFF-45 (DNA fragmentation factor) degradation. These results lead to a working hypothesis that penta- O-galloyl-β- d-glucose-induced apoptosis is triggered by the release of cytochrome c into the cytosol, procaspase-9 processing, activation of caspase-3, degradation of poly-(ADP-ribose) polymerase, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by penta- O-galloyl-β- d-glucose may provide a pivotal mechanism for its cancer chemopreventive action.

Disaccharide esters screened for inhibition of tumor necrosis factor-alpha release are new anti-cancer agents.

Tumor necrosis factor‐α (TNF‐α) is a proinflammatory cytokine playing a part in various pathological states. Non‐toxic inhibitors of TNF‐α release are thought to be promising agents for cancer prevention. We found that the acetone fraction of the tobacco leaf surface lipid containing glucose esters and sucrose esters inhibited both TNF‐α release from BALB/3T3 and KATO III cells induced by okadaic acid and tumor promotion by okadaic acid on mouse skin initiated with 7,12‐dimethylbenz(a)anthracene (DMBA). Next, we investigated the inhibition of TNF‐α release with synthetic disaccharide esters, such as 6,6′‐di‐0‐alkanoyl‐α,α‐trehaloses (6,6′‐diester‐trehaloses), 4,4′‐di‐0‐alkanoyl‐α,α‐trehaloses (4,4′‐diester‐trehaloses) and 6,6′‐diamino‐6,6′‐dideoxy‐N, N′‐dial‐kanoyl‐α,α‐trehaloses (6,6′‐diamide‐trehaloses) bearing fatty acids of various chain lengths, and n‐ dodecyl‐β‐D‐maltoside as a disaccharide monoester. 6,6′‐Diester‐trehaloses and 4,4′‐diester‐treha‐loses of C8 to C14 fatty acids, 6,6′‐diamide‐trehaloses of C8 to C14 fatty acids, and n‐dodecyl‐β‐D‐maltoside all inhibited TNF‐α release in a dose‐dependent manner. The IC50 values are 7.4‐14.8 μM for 6,6′‐diester‐trehaloses (C8 to C12), 14.6‐21.6 μM 4,4′‐diester‐trehaloses (C8 to C12), 2.9‐15.0 μM for 6,6′‐diamide‐trehaloses (C8 to C14) and 23 μM for dodecyl‐β‐D‐maltoside. Both 6,6′‐di‐O‐octanoyl‐α,α‐trehalose (C8, designated as SS555) and n‐dodecyl‐β‐D‐maltoside (C12) inhibited tumor promotion by okadaic acid on mouse skin initiated with DMBA. Percentages of tumor‐bearing mice in week 15 of tumor promotion were reduced from 60.0 to 13.3 with SS555, and to 46.7 with n‐dodecyl‐β‐D‐maltoside. Moreover, SS555 inhibited TNF‐α gene expression mediated through inhibition of AP‐1 activation, but not NF‐αB activation. This paper reports that diester‐trehaloses of C8 to C12 fatty acids and mimics of disaccharide monoesters such as n‐dodecyl‐β‐D‐maltoside appear to be potential cancer‐preventive agents of a new type.

Mechanistic findings of green tea as cancer preventive for humans.

Based on our initial work with green tea, in which repeated topical applications of (-)-epigallocatechin gallate (EGCG), the main green tea polyphenol, inhibited tumor promotion in a two-stage carcinogenesis experiment on mouse skin (Phytother Res 1, 44-47, 1987), numerous scientists have since provided so much additional evidence of the benefits of drinking green tea that it is now an acknowledged cancer preventive in Japan, and will possibly soon be recognized as such in other countries. Our work has so far produced several important results with EGCG and green tea: a wide range of target organs in animal experiments for cancer prevention, wide bioavailability of 3H-EGCG in various organs of mice, delayed cancer onset of patients with a history of consuming over 10 cups of green tea per day, and absence of any severe adverse effects among volunteers who took 15 green tea tablets per day (2.25 g green tea extracts, 337.5 mg EGCG, and 135 mg caffeine) for 6 months. This paper introduces three new findings: 1) EGCG interacted with the phospholipid bilayer membrane resulting in confirmation of the sealing effect of EGCG; 2) EGCG inhibited TNF-alpha gene expression in the cells and TNF-alpha release from the cells; 3) high consumption of green tea was closely associated with decreased numbers of axillary lymph node metastases among premenopausal Stage I and II breast cancer patients, and with increased expression of progesterone and estrogen receptors among postmenopausal ones. These results provide new insights into our understanding of the mechanisms of action of tea polyphenols and green tea extract as a cancer preventive.

Structurally Novel Bioconversion Products of the Marine Natural Product Sarcophine Effectively Inhibit JB6 Cell Transformation.

Sarcophytol A (1) and B (2) (see Chart 1) are cembrane-type diterpenes known to inhibit tumor promotion. Indicative of this inhibitory response, we currently report sarcophytol A (1) mediates dose-dependent diminution of 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced transformation of JB6 cells. Moreover, a structurally related furanocembrane diterpene, sarcophine (3), isolated in good yield from the Red Sea soft coral Sarcophyton glaucum, was also found to serve as an effective inhibitor of JB6 cell transformation. This compound was subjected to preparative-scale fermentation with Absidia glauca ATCC 22752, Rhizopus arrhizus ATCC 11145, and Rhizopus stolonifer ATCC 24795, resulting in the production of 10 new metabolites (5-14) along with the known compound 7beta,8alpha-dihydroxydeepoxysarcophine (4). Structures were elucidated primarily on the basis of 2D-NMR spectroscopy, with X-ray crystallography being used to establish the relative stereochemistry of metabolite 5. When evaluated for potential to inhibit TPA-induced JB6 cell transformation, several of the metabolites mediated inhibitory responses greater than sarcophytol A (1) or sarcophine (3), most notably 7alpha-hydroxy-Delta(8(19))-deepoxysarcophine (6), which was comparable to 13-cis-retinoic acid. These studies provide a basis for further development of novel furanocembranoids as anticancer agents.

Suppression of mouse skin tumor promotion and induction of apoptosis in HL-60 cells by Alpinia oxyphylla Miquel (Zingiberaceae).

There have been considerable efforts to search for naturally occurring substances for the intervention of carcinogenesis. Many components from dietary or medicinal plants have been identified that possess substantial chemopreventive properties. An example is curcumin (Curcuma longa Linn., Zingiberaceae), which has been shown to inhibit tumor promotion in experimental carcinogenesis. Alpinia oxyphylla Miquel, another plant of the ginger family used in oriental herbal medicine, contains diarylheptanoids whose structures are analogous to that of curcumin. In the present study, we have tested A.oxyphylla for its ability to suppress tumor promotion. Thus, topical application of the methanolic extract of dried fruits of A.oxyphylla significantly ameliorated 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumor promotion as well as ear edema in female ICR mice. In another study, treatment of HL-60 cells with the methanolic extract of A.oxyphylla significantly reduced the viability of the cells and also inhibited DNA synthesis. Microscopic examination of the treated cells showed characteristic morphology of apoptosis. Furthermore, cells treated with the extract of A.oxyphylla exhibited internucleosomal DNA fragmentation in time- and concentration-dependent manners. TPA-stimulated generation of superoxide anion in differentiated HL-60 cells was also blunted by A.oxyphylla. Taken together, these findings suggest that A.oxyphylla possesses potential chemopreventive and antitumorigenic activities.

Inhibitory effect of Pruni Cortex extract and its component, octacosyl ferulate, on tumour promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin

The methanol extract of Pruni Cortex inhibited tumour promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mice. The active component was isolated from the methanol extract of Pruni Cortex. The active compound was characterized as octacosyl ferulate by physical and chemical analysis. Octacosyl ferulate markedly inhibited the inflammatory activity induced by TPA in mice. The 50% inhibitory dose of octacosyl ferulate is 0.7 mg/ear on TPA-induced inflammation. Furthermore, octacosyl ferulate suppressed markedly the tumour-promoting effect of TPA on skin tumour formation following initiation with DMBA. Since protein kinase C (PK-C) was shown to be an intracellular target of TPA, effects of octacosyl ferulate on the activity of this enzyme were investigated. Octacosyl ferulate also inhibited the phosphorylation of histon III-S by PK-C in a concentration dependent manner. © 1998 John Wiley & Sons, Ltd.

Selenocompounds Induce a Redox Modulation of Protein Kinase C in the Cell, Compartmentally Independent from Cytosolic Glutathione: Its Role in Inhibition of Tumor Promotion

Since selenite and other redox-active selenocompounds can modify protein kinase C (PKC) in the test tube, we have determined whether or not this redox regulation occurs inside the cell despite having high concentrations of GSH and the role of this regulation in the inhibition of tumor promotion. By using phorbol ester-promoted JB6 epidermal cell transformation assay, the concentrations of selenite, selenocystine, and selenodiglutathione which are optimal for chemopreventive activity were determined. At such concentrations (0.5 to 2 μm) in the cells treated with these agents, only a slight but transient decrease in PKC activity was observed when measured with a low (5 μm), but not with a high (100 μm) concentration of ATP. However, when the cells were serum starved or pretreated with 2-deoxyglucose, there was a pronounced but transient inactivation of PKC when assayed with both low and high concentrations of ATP. The inactivation was reversed in the cell by an endogenous mechanism or by treatment with thiol agents in the test tube. In spite of a substantial (90%) depletion of GSH in the cells by pretreatment with buthionine sulfoximine, there was no further increase in the redox modification of PKC by selenite as well as no change in the inhibitory effect of selenite on the phorbol ester-stimulated induction of ornithine decarboxylase, which is an intermediate marker related to cell transformation. While GSH is known to influence certain actions of selenium, it may not be required to mediate the effects of selenite tested in this study. The water-soluble cytosolic GSH did not interfere with the redox modification of PKC probably due to the shielding of the cysteine-rich region of the enzyme by a weak hydrophobic association with the membrane. Due to the presence of cofactors in the crude cell extracts, PKC was more sensitive to selenite than in the purified form and was inactivated by low concentrations of selenite (IC50= 0.05 μm). This modification was reversed by thiol agents as well as by NADPH. A protein disulfide reductase, which can regenerate PKC, was present in the homogenate. Conceivably, selenite and other selenocompounds induce a redox modification of cellular PKC, compartmentally independent from the cytosolic GSH, but intimately connected to a NADPH-dependent reductase system, to mediate, at least in part, some of the cancer-preventive actions.

Inhibition of tumour promoter-induced Epstein- Barr virus activation by diospyrin, a plant-derived antitumour compound, and its synthetic derivatives

Inhibition of tumour promotion by diospyrin, a plant-derived bioactive bisnaphthoquinonoid compound, and its two derivatives was examined by an in vitro assay utilizing the activation of Epstein–Barr virus (EBV) early antigen (EA) expression in EBV- genome- carrying human lymphoblastoid cells. Synthetic modifications of diospyrin resulted in positive improvement of its activity against tumour promotion by inhibiting EBV EA expression in Raji cells induced by 12-o-tetradecanoylphorbol-13-acetate (TPA). © 1997 John Wiley & Sons, Ltd.

Auraptene, a citrus coumarin, inhibits 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion in ICR mouse skin, possibly through suppression of superoxide generation in leukocytes.

Coumarin‐related compounds, auraptene and umbelliferone, have been isolated from the cold‐pressed oil of natsumikan (Citrus natsudaidai HAYATA), and tested as inhibitors of tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced Epstein‐Barr virus activation in Raji cells. The 50% inhibitory concentration (IC50) of auraptene (18μM)was almost equal to that of genistein. Umbelliferone, which lacks a geranyloxyl group present in auraptene, was less active (IC50=450 μM). In a two‐stage carcinogenesis experiment with 7, 12‐dimethylbenz[α]anthracene (topical application at 0.19 μmol) and TPA (topical application at 1.6 nmol) in ICR mouse skin, topical application of auraptene (at 160 nmol) significantly reduced tumor incidence and the numbers of tumors per mouse by 27% (P < 0.01) and 23% (P < 0.05), respectively. Auraptene at a concentration of 50 μM markedly suppressed superoxide (O2−) generation induced by 100 nM TPA in differentiated human promyelocytic HL‐60 cells. Having no O2− ‐scavenging potential, auraptene may inhibit the multicomponent NADPH oxidase system. Inhibition of intracellular hydroperoxide formation in differentiated HL‐60 cells by auraptene was also confirmed by flow‐cytometric analysis using 2′,7′‐dichlorofluorescein diacetate as a fluorescence probe. Quantitative analyses using high‐performance liquid chromatography showed the occurrence of auraptene not only in both the peels and sarcocarps of natsumikan, but also in those of hassaku orange (C. hassaku) and grapefruit (C. paradisi,) and even in their bottled fresh juice form. These results indicate that auraptene is a chemopreventer of skin tumorigenesis, and implies that suppression of leukocyte activation might be the mechanism through which it inhibits tumor promotion.

Anti-tumor promoting activity of canventol and its synthetic analogs through inhibition of protein isoprenylation.

Canventol, a synthetic compound, is a new inhibitor of tumor promotion on mouse skin by okadaic acid. We previously reported that canventol acts by inhibiting both protein isoprenylation and tumor necrosis factor‐α (TNF‐α) release. In this study we examined the potencies of 10 newly synthesized canventol analogs through their effect on mevalonate metabolism, and then examined 3 representative analogs for inhibition of protein isoprenylation. Since canventol in vitro did not directly inhibit farnesyl protein transferase or geranylgeranyl protein transferase‐I, the effects of canventol and its synthetic analogs on the fate of [3H]mevalonate in cells were studied. Canventol at 500 μM changed the ratio of newly synthesized sterols (cholesterol and lathosterol) to ubiquinones from 0.7 to 8.2 in NIH/3T3 cells which had previously been labeled with [3H]mevalonate, suggesting that the altered pattern of mevalonate metabolism is associated with inhibition of protein isoprenylation in the cells. We named this ratio the inhibition of protein isoprenylation index (IPI index). The 10 analogs showed a wide range of IPI indices. Two analogs, S3 and S9 had effects similar to, or stronger than, canventol. Three analogs, C44, C46 and C47, with lower IPI indices, inhibited tumor promotion on mouse skin slightly less than canventol itself did. This study shows that inhibition of protein isoprenylation in the cells, indicated by an increase in the IPI index, is a new biomarker for estimating inhibition of tumor promotion.

Inhibitory Effect of Methanol Extracts from Edible Mushroom on TPA‐induced Ear Oedema and Tumour Promotion in Mouse Skin

Edible mushroom extracts inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear oedema in mice. Twenty seven methanol extracts obtained from mushrooms were assayed and their inhibition ratios calculated. The methanol extracts of Russula delica, Mycoleptodonoides aitchisonii, Lactarius deliciosus, Naematoloma sublateritium, Lyophyllum decastes and Lyophyllum connatum markedly inhibited the inflammatory activity induced by TPA in mice. The methanol extract from Mycoleptodonoides aitchisoni was found to inhibit tumour promotion in two-stage carcinogenesis in mouse skin.

Inhibitory Effect of Methanol Extracts from Edible Mushroom on TPA-induced Ear Oedema and Tumour Promotion in Mouse Skin

Edible mushroom extracts inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear oedema in mice. Twenty seven methanol extracts obtained from mushrooms were assayed and their inhibition ratios calculated. The methanol extracts of Russula delica, Mycoleptodonoides aitchisonii, Lactarius deliciosus, Naematoloma sublateritium, Lyophyllum decastes and Lyophyllum connatum markedly inhibited the inflammatory activity induced by TPA in mice. The methanol extract from Mycoleptodonoides aitchisoni was found to inhibit tumour promotion in two-stage carcinogenesis in mouse skin.

Inhibition of tumor promotion and hepatocellular growth by dietary restriction in mice.

The effects of dietary restriction on the growth of hepatic focal lesions in phenobarbital (PB) promoted mice were examined. Dietary restriction which can inhibit many age-related diseases in rodents including hepatic cancer also decreases cell proliferation and increases apoptosis in the liver. In contrast, PB, a non-genotoxic rodent hepatocarcinogen, enhances the growth of hepatic focal lesions in mice and rats by increasing cell proliferation and inhibiting apoptosis. The present study examined the impact of dietary restriction on PB-induced hepatic tumor promotion. Focal lesions were produced by diethylnitrosamine (DEN) treatment (35 mg DEN/kg body weight injections, twice per week for 8 weeks). After lesions were produced, mice were placed into one of the following four groups: NIH-07 control diet/no PB (group 1); NIH-07 diet/500 mg PB per liter of drinking water (group 2); dietary restricted NIH-07 diet/no PB (group 3); and dietary restricted NIH-7 diet/ 500 mg PB per liter of drinking water (group 4). In this study, PB (500 mg/l) treatment to ad libitum-fed mice (group 2) enhanced focal lesion volume, number, and labeling index compared with group 1. In addition, PB treatment (group 2) inhibited apoptosis in normal and focal hepatocytes compared with untreated control mice (group 1). In contrast, in dietary restricted mice treated with PB (group 4) a significantly lower focal lesion volume, number and labeling index were seen compared with the ad libitum-fed/PB treatment group (group 2). PB treatment in dietary restricted mice (group 4) did not inhibit focal apoptosis, in fact, the incidence of focal apoptosis was increased in these mice compared with ad libitum and PB-treated mice (group 2). In dietary restricted mice treated with PB (group 4), the ability of PB to promote the growth of preneoplastic focal lesions was inhibited. These results show that dietary restriction can ablate the tumor promotional effects of PB in hepatic focal lesions and suggest that inhibition of focal lesion DNA synthesis and enhancement of apoptosis may be a mechanism for this effect.

Anti-tumor promotion with food phytochemicals: a strategy for cancer chemoprevention.

Cancer chemoprevention is currently regarded as a promising avenue for cancer control. In particular, the inhibition of tumor promotion (anti-tumor promotion) in multistage carcinogenesis is expected to be an efficient strategy, because tumor promotion is experimentally accomplished through the long-term, repetitive exposures of rodents to a tumor promoter, and premalignant lesions caused by a tumor promoter regress, at least in their earlier stages. In this review, we first describe the background of cancer chemoprevention studies as well as recent results of clinical trials. Subsequently, some hypothetical biological and cellular pathways in tumor promotion are explored. In addition, the anti-tumor promoting properties of vegetables, fruits, and edible marine algae, together with their active constituents and action mechanisms thus far known, are also described. Anti-tumor promotion with food phytochemicals may be characterized as an efficient and reliable strategy for cancer chemoprevention.

Naturally-Derived Tumor Promoters and Inhibitors of Carcinogenesis

Tumor promoters are agents that develop tumors from initiated cells, and tumor promotion is an important stage in multi-stage carcinogenesis in humans. We studied mechanisms of tumor promotion in two-stage carcinogenesis experiments in rodents with tumor promoters derived from various marine organisms. The compounds of the lyngbyatoxin and aplysiatoxin classes, like 12–O-tetradecanoylphorbl-13-acetate (TPA) derived from Euphorbia plants, induced tumor promotion on mouse skin mediated through activation of protein kinase C, and were identified as the TPA-type tumor promoters. The non-TPA type tumor promoters included the palytoxin and okadaic acid classes. The okadaic acid class compounds induced tumor promotion in various organs mediated through inhibition of protein phosphatases 1 and 2A. The study of the okadaic acid class of tumor promoters led us to find tumor necrosis factor-α as a tumor promoter endogenously present in human tissues. Inhibitors of tumor promotion were also found in marine organisms, such as sarcophytol A and discodermin A. In addition, agelasine-B was found to be a new activator of protein phosphatases 1 and 2A. This paper reviews our study on tumor promotion with marine toxins and presents an essential mechanism of tumor promotion in human cancer development.

1'-Acetoxychavicol acetate, a superoxide anion generation inhibitor, potently inhibits tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in ICR mouse skin.

The anti-tumor-promoting activity of 1'-acetoxychavicol acetate (ACA) was examined in a two-stage carcinogenesis experiment in ICR mouse skin using 7,12-dimethylbenz[a]anthracene (0.19 mumol) and 12-O-tetradecanoylphorbol-13-acetate (TPA; 1.6 nmol). Topical application of ACA (160 nmol) markedly reduced the average number of tumors per mouse and the ratio of tumor-bearing mice: inhibition ratios 90% (p < 0.001) and 42% (p < 0.005), respectively. ACA even at a dose equimolar to TPA (1.6 nmol) significantly reduced the average number of tumors per mouse: inhibitory ratio 44% (p < 0.05). ACA potently inhibited TPA-induced superoxide (O2-) generation in differentiated HL-60 cells (IC50 = 4.3 microM) and suppressed the lipid hydroperoxide formation by 42% (p < 0.001) in the ethyl linoleate autoxidation test.

Dietary conjugated linoleic acid modulation of phorbol ester skin tumor promotion.

The fatty acid derivative conjugated dienoic linoleate (CLA) has been shown to inhibit initiation and postinitiation stages of carcinogenesis in several experimental animal models. The goal of the present study was to determine the role of increasing levels of dietary CLA in mouse skin tumor promotion elicited by 12-O-tetradecanoylphorbol-13-acetate (TPA). Mice were fed control (no CLA) diet during initiation, then switched to diets containing 0.0%, 0.5%, 1.0%, or 1.5% (wt/wt) CLA during skin tumor promotion by TPA. Body weights of mice fed 0.5%, 1.0%, or 1.5% CLA were similar to each other but were significantly lower (p < 0.05) than weights of mice fed no CLA (0.0%) throughout promotion. A reduction in papilloma incidence was observed in mice fed 1.5% CLA from Weeks 8 to 24 compared with mice fed diets containing 0.0-1.0% CLA (p < 0.05). Twenty-four weeks after tumor promotion was begun, diets containing 1.0% and 1.5% CLA inhibited tumor yield (4.94 and 4.35 tumors/mouse, respectively) compared with diets without CLA (0.0% CLA, 6.65 tumors/mouse, p < 0.05) or 0.5% CLA (5.92 tumors/mouse, p < 0.05). These data indicate that CLA inhibits tumor promotion in a manner that is independent of its anti-initiator activity. Further studies are warranted in identifying cellular mechanisms that are likely to be involved with the antipromoter effects of CLA.

Suppression by carotenoids of microcystin-induced morphological changes in mouse hepatocytes.

Microcystin-LR is a liver tumor promoter in the okadaic acid class, a group of potent inhibitors of protein phosphatases 1 and 2A. Because of inhibition of protein phosphatases, microcystin-LR induces hyperphosphorylation of cellular proteins, including cytoskeletal proteins--cytokeratins 8 and 18--and causes morphological changes in mouse hepatocytes in primary culture. We studied the effects of carotenoids to antagonize microcystin-LR-induced morphological changes in hepatocytes. beta-carotene (100 nM to 100 microns) suppressed the morphological changes induced by 100 nM microcystin-LR in a dose-dependent manner. Other carotenoids tested exerted similar suppressive effects, although retinoids, such as all-trans retinol, all-trans retinoic acid, and 9-cis retinoic acid, were only weakly suppressive. The relative potency of the suppression correlated significantly with the number of conjugated double bonds in the trans configuration. beta-carotene strongly suppressed the hyperphosphorylation of cellular proteins induced by microcystin-LR without significant changes in the basal phosphorylation level. Other antioxidants, such as alpha-tocopherol, did not protect the cells against microcystin-LR. Taken together, the antagonistic effects of carotenoids against microcystin-LR are difficult to explain by their antioxidant or provitamin A activities. Suppression of the hyperphosphorylation of cellular proteins may be a novel mechanism by which carotenoids inhibit tumor promotion.

Alkane-6,8-diol: inhibitor of tumor promotion in two-stage carcinogenesis in mouse skin.

Alkane-6,8-diol: inhibitor of tumor promotion in two-stage carcinogenesis in mouse skin.